A. Orthopoxviruses
The poxviruses comprise a large family of complex DNA viruses that infect both vertebrate and invertebrate hosts. General properties of the Poxvirus family include (a) a large complex virion containing enzymes for mRNA synthesis, (b) a genome composed of a single linear double-strand DNA molecule of 130 to 300 kilobases, and (c) the ability to replicate within the cytoplasmic compartment of the cell. The vertebrate poxviruses have been placed into six genera: Orthopoxvirus, Parapoxvirus, Capripoxvirus, Leporipoxvirus, Suipoxvirus, and Avipoxvirus. 
Three members of the Orthopoxvirus genus are known to cause disease in humans. The most notorious member of the Poxvirus family is the variola virus which, before its eradication, was responsible for smallpox. Cowpox virus and Monkeypox virus also cause disease in humans. Additional members of the Orthopoxvirus genus include: Buffalopox virus, Camelpox virus, Rabbitpox virus, Raccoonpox virus, Volepox virus and Ectromeila virus.
B. Bioagent Detection
A problem in determining the cause of a natural infectious outbreak or a bioterrorist attack is the sheer variety of organisms that can cause human disease. There are over 1400 organisms infectious to humans; many of these have the potential to emerge suddenly in a natural epidemic or to be used in a malicious attack by bioterrorists (Taylor et al., Philos. Trans. R. Soc. London B. Biol. Sci., 2001, 356, 983-989). This number does not include numerous strain variants, bioengineered versions, or pathogens that infect plants or animals.
Much of the new technology being developed for detection of biological weapons incorporates a polymerase chain reaction (PCR) step based upon the use of highly specific primers and probes designed to selectively detect individual pathogenic organisms. Although this approach is appropriate for the most obvious bioterrorist organisms, like smallpox and anthrax, experience has shown that it is very difficult to predict which of hundreds of possible pathogenic organisms might be employed in a terrorist attack. Likewise, naturally emerging human disease that has caused devastating consequence in public health has come from unexpected families of bacteria, viruses, fungi, or protozoa. Plants and animals also have their natural burden of infectious disease agents and there are equally important biosafety and security concerns for agriculture.
An alternative to single-agent tests is to perform broad-range consensus priming of a gene target conserved across groups of bioagents. Broad-range priming has the potential to generate amplification products across entire genera, families, or, as with bacteria, an entire domain of life. This strategy has been successfully employed using consensus 16S ribosomal RNA primers for determining bacterial diversity, both in environmental samples (Schmidt et al., J. Bact., 1991, 173, 4371-4378) and in natural human flora (Kroes et al., Proc. Nat. Acad. Sci. (USA), 1999, 96, 14547-14552). One drawback of this approach for unknown bioagent detection and epidemiology is that analysis of the PCR products requires cloning and sequencing of hundreds to thousands of colonies per sample, which is impractical to perform rapidly or on a large number of samples.
Conservation of sequence is not as universal for viruses. Large groups of viral species, however, share conserved protein-coding regions, such as regions encoding viral polymerases or helicases. Like bacteria, consensus priming has also been described for detection of several viral families, including coronaviruses (Stephensen et al., Vir. Res., 1999, 60, 181-189), enteroviruses (Oberste et al., J. Virol., 2002, 76, 1244-51; Oberste et al., J. Clin. Virol., 2003, 26, 375-7; and Oberste et al., Virus Res., 2003, 91, 241-8), retroid viruses (Mack et al., Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6977-81; Seifarth et al., AIDS Res. Hum. Retroviruses, 2000, 16, 721-729; and Donehower et al., J. Vir. Methods, 1990, 28, 33-46), and adenoviruses (Echavarria et al., J. Clin. Micro., 1998, 36, 3323-3326). However, as with bacteria, there is no adequate analytical method other than sequencing to identify the viral bioagent present.
In contrast to PCR-based methods, mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated. DNA chips with specific probes can only determine the presence or absence of specifically anticipated organisms. Because there are hundreds of thousands of species of benign pathogens, some very similar in sequence to threat organisms, even arrays with 10,000 probes lack the breadth needed to identify a particular organism.
There is a need for a method for identification of bioagents which is both specific and rapid, and in which no culture or nucleic acid sequencing is required.
The present invention provides, inter alia, methods of identifying unknown viruses, including viruses of the Orthopoxvirus genus. Also provided are oligonucleotide primers, compositions, and kits containing the oligonucleotide primers, which define orthopoxvirus identifying amplicons and, upon amplification, produce corresponding amplification products whose molecular masses provide the means to identify orthopoxviruses at the species and sub-species or strain level.